Method for diagnosing preeclampsia by detecting hCRH mRNA

ABSTRACT

Methods and kits are provided for diagnosing, monitoring, or predicting the conditions of pre-eclaimpsia, fetal chromosomal aneuploidy, and pre-term labor in a pregnant woman, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more mRNA species encoding human chorionic gonadotropin β subunit (hCG-β), human placental lactogen (hPL), human corticotropin releasing hormone (hCRH), KiSS-1 metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2 (TPFI2), placenta-specific 1 (PLAC1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and comparing the amount of the mRNA species with a standard control.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60,440,906, filed Jan. 17, 2003, the content of which isincorporated herein by reference in the entirety.

BACKGROUND OF THE INVENTION

Prenatal diagnosis has been routinely conducted using cells isolatedfrom the fetus through procedures such as chorionic villus sampling(CVS) or amniocentesis. These conventional methods are, however,invasive and present an appreciable risk to both the mother and thefetus despite most careful handling (Tabor et al., Lancet 1:1287–1293,1986).

Alternatives to these invasive approaches have been developed forprenatal screening, e.g., to detecting fetal abnormalities, followingthe discoveries that several types of fetal cells can be found inmaternal circulation (Johansen et al., Prenat. Diagn. 15:921–931, 1995)and more importantly, circulating cell-free fetal DNA can be detected inmaternal plasma and serum (Lo et al., Lancet 350:485–487, 1997). Theamount of fetal DNA in maternal blood has been shown to be sufficientfor genetic analysis without complex treatment of the plasma or serum,in contrast to the necessary steps for isolating and enriching fetalcells. Fetal rhesus D (RhD) genotyping (Lo et al., N. Engl. J. Med.339:1734–1738, 1998), fetal sex determination (Lo et al., Hum. Genet.90:483–488, 1993), and diagnosis of several fetal disorders (Amicucci etal., Clin. Chem. 46:301–302, 2000; Saito et al., Lancet 356:1170, 2000;and Chiu et al., Lancet 360:998–1000, 2002) have since been achieved bydetecting fetal DNA in maternal blood using a polymerase chain reaction(PCR)-based technique.

In addition, quantitative abnormalities of fetal DNA in maternalplasma/serum have also been reported in preeclampsia (Lo et al., Clin.Chem. 45:184–188, 1999 and Zhong et al., Am. J. Obstet. Gynecol184:414–419, 2001), fetal trisomy 21 (Lo et al., Clin. Chem.45:1747–1751, 1999 and Zhong et al. Prenat. Diagn. 20:795–798, 2000) andhyperemesis gravidarum (Sekizawa et al., Clin. Chem. 47:2164–2165,2001). Detection of fetal nucleic acid in maternal blood for prenatalgenetic analysis is also disclosed in U.S. Pat. No. 6,258,540.

When analyzing fetal DNA, investigators have often used Y chromosomalmarkers, which are only present in male fetuses, as a fetal-specificmarker. This approach has limited the application of this technology tothe 50% of pregnant women who are carrying male fetuses. Further, theuse of other genetic polymorphisms has also increased the complexity offetal DNA-based analyses. The discovery of fetal RNA in maternal plasmaoffers a possible new approach that circumvents these limitations (Poonet al., Clin. Chem. 46:1832–1834, 2000).

More recently, U.S. patent application Ser. No. 09/876,005 disclosesnon-invasive techniques based on detection of fetal RNA in maternalblood. The present invention discloses for the first time that theamount of certain mRNA species present in maternal blood, includingthose encoding human chorionic gonadotropin β subunit (hCG-β), humancorticotropin releasing hormone (hCRH), human placental lactogen (hPL),KiSS-1 metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2(TPFI2), placenta-specific 1 (PLAC1), or glyceraldehyde-3-phosphatedehydrogenase (GAPDH), can be used as markers for diagnosing,monitoring, or predicting pregnancy-related disorders such aspreeclampsia, fetal chromosomal aneuploidy, and pre-term labor, as wellas for detecting pregnancy.

BRIEF SUMMARY OF THE INVENTION

The present invention provides new methods for diagnosing, monitoring,or predicting pregnancy-related disorders such as preeclampsia, fetalchromosomal aneuploidy, and pre-term labor by measuring the amount ofone or more species of circulating mRNA found in the maternal blood.Methods for detecting pregnancy in a woman are also provided based onthe same methodology. The mRNA may encode proteins of fetal or maternalorigin such as human chorionic gonadotropin β subunit (hCG-β), humanplacental lactogen (hPL), human corticotropin releasing hormone (hCRH),KiSS-1 metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2(TPFI2), placenta-specific 1 (PLAC1), and glyceraldehyde-3-phosphatedehydrogenase (GAPDH). Diagnostic/detection kits for thesepregnancy-related conditions/pregnancy based on the invention are alsoprovided.

One aspect of the present invention relates to a method for diagnosing,monitoring, or predicting preeclampsia in a pregnant woman. This methodcomprises multiple steps: the first step is to quantitatively determinethe amount of one or more particular species of mRNA present in thepregnant woman's blood. The mRNA may encode hCG-β, hCRH, hPL, KISS1,TPFI2, PLAC1, or GAPDH. The second step is to compare the amount of mRNAobtained from the first step to a standard control representing theamount of mRNA encoding the same protein(s) in the blood of an averagenon-preeclamptic woman. An increase or a decrease in the mRNA levelindicates either the presence of preeclampsia or an increased risk ofdeveloping the condition.

In some embodiments, the mRNA used as marker in this method encodes hCRHor GAPDH. In some embodiments, the first step of the method is performedby reverse transcription polymerase chain reaction (RT-PCR), whereas inothers this first sleep is carried out using a polynucleotidehybridization method or mass spectrometry. In some embodiments, thepregnant woman is during the first trimester of gestation, whereas inothers the woman is during the second or third trimester of gestation.In some embodiments, the pregnant woman's blood is rendered acellularbefore it is used in the first step of the method. In some embodiments,plasma or serum is used in the first step of the method. In someembodiments, the increase in the amount of mRNA from the standardcontrol is more than 2-fold.

The method of the present invention may also be used for diagnosis,monitoring, or prediction of a more severe clinical course thanpreeclampsia, such as eclampsia.

The invention also relates to a kit for diagnosing, monitoring, orpredicting preeclampsia in a pregnant woman. The kit comprises PCRprimers for quantitatively determining the amount of one or moreparticular species of mRNA in the pregnant woman's blood. The mRNA mayencode hCG-β, hCRH, hPL, KISS1, TPFI2, PLAC1, or GAPDH. The kit furthercomprises a standard control that represents the amount of mRNA encodingthe same protein(s) in an average non-preeclamptic pregnant woman. A kitof the present invention may, in addition to or in place of the PCRprimers, comprise one or more probes that can be used to quantitativelydetermine the amount of mRNA encoding hCG-β, hCRH, hPL, KISS1, TPFI2,PLAC1, or GAPDH.

Another aspect of the present invention relates to a method fordetecting the presence of a fetus with a chromosomal aneuploidy, such astrisomy 18 or trisomy 21, in a pregnant woman. This method comprisesmultiple steps: the first step is to quantitatively determine the amountof one or more particular species of mRNA present in the pregnantwoman's blood. The mRNA may encode hCG-β, hCRH, hPL, KISS1, TPFI2,PLAC1, or GAPDH. The second step is to compare the amount of mRNAobtained from the first step to a standard control representing theamount of mRNA encoding the same protein(s) in the blood of an averagepregnant woman carrying a chromosomally normal fetus. An increase or adecrease in the mRNA level indicates an increased risk of having ananeuploid fetus.

In some embodiments, the mRNA used as a marker in the method encodeshCG-β. In some embodiments, the first step of the method is performed byRT-PCR, whereas in others this first step is carried out using apolynucleotide hybridization method or mass spectrometry. In someembodiments, the pregnant woman is during the first trimester ofgestation, whereas in others the woman is during the second or thirdtrimester of gestation. In some embodiments, the pregnant woman's bloodis rendered acellular before it is used in the first step of the method.In some embodiments, plasma or serum is used in the first step of themethod. In some embodiments, the increase is at least 2-fold. In otherembodiments, the decrease is at lease 50%.

The invention also relates to a kit for detecting the presence of afetus with a chromosomal aneuploidy (such as trisomy 18 or trisomy 21)in a pregnant woman. The kit comprises PCR primers for quantitativelydetermining the amount of one or more particular species of mRNA in thepregnant woman's blood. The mRNA may encode hCG-β, hCRH, hPL, KISS1,TPFI2, PLAC1, or GAPDH. The kit further comprises a standard controlthat represents the amount of mRNA encoding the same protein(s) in anaverage pregnant woman carrying a chromosomally normal fetus. A kit ofthe present invention may, in addition to or in place of the PCR primes,comprise one or more probes that can be used to quantitatively determinethe amount of mRNA encoding hCG-β, hCRH, hPL, KISS1, TPFI2, PLAC1, orGAPDH.

A further aspect of the present invention relates to a method fordiagnosing, monitoring, or predicting pre-term labor in a pregnantwoman. This method comprises multiple steps: the first step is toquantitatively determine the amount of one or more particular species ofmRNA present in the pregnant woman's blood. The mRNA may encode hCG-β,hCRH, hPL, KISS1, TPFI2, PLAC1, or GAPDH. The second step is to comparethe amount of mRNA obtained from the first step to a standard controlrepresenting the amount of mRNA encoding the same protein(s) in theblood of an average woman who delivers or will deliver at term. Anincrease or a decrease in the mRNA level indicates pre-term labor or anincreased risk of developing the condition.

In some embodiments, the first step of the method is performed byRT-PCR, whereas in others this first step is carried out using apolynucleotide hybridization method or mass spectrometry. In someembodiments, the pregnant woman is during the first trimester ofgestation, whereas in others the woman is during the second or thirdtrimester of gestation. In some embodiments, the pregnant woman's bloodis rendered acellular before it is used in the first step of the method.In some embodiments, plasma or serum is used in the first step of themethod. In some embodiments, the increase in the amount of mRNA from thestandard control is more than 2-fold. In other embodiments, the decreaseis at least 50%.

The invention also relates to a kit for diagnosing, monitoring, orpredicting pre-term labor in a pregnant woman. The kit comprises PCRprimers for quantitatively determining the amount of one or moreparticular species of mRNA in the pregnant woman's blood. The mRNA mayencode hCG-0, hCRH, hPL, KISS1, TPFI2, PLAC1, or GAPDH. The kit furthercomprises a standard control that represents the amount of mRNA encodingthe same protein(s) in an average pregnant woman who delivers or willdeliver at term. A kit of the present invention may, in addition to orin place of the PCR primes, comprise one or more probes that can be usedto quantitatively determine the amount of mRNA encoding hCG-β, hCRH,hPL, KISS1, TPFI2, PLAC1, or GAPDH.

An additional aspect of the invention relates to a method for detectingpregnancy in a woman. This method comprises multiple steps: the firststep is to quantitatively determine the amount of one or more particularspecies of mRNA present in the woman's blood. The mRNA may encodehCG-fl, hCRH, hPL, KISS1, TPFI2, or PLAC1. The second step is to comparethe amount of mRNA obtained from the first step to a standard controlrepresenting the amount of mRNA encoding the same protein(s) in theblood of an average woman who is healthy and not pregnant. An increasein the mRNA level indicates pregnancy.

In some embodiments, the first step of the method is performed byRT-PCR, whereas in others this first step is carried out using apolynucleotide hybridization method or mass spectrometry. In someembodiments, the woman's blood is rendered acellular before it is usedin the first step of the method. In some embodiments, plasma or serum isused in the first step of the method. In some embodiments, the increasein the amount of mRNA from the standard control is more than 2-fold. Inother embodiments, the decrease is at least 50%.

The invention also relates to a kit for detecting pregnancy in a woman.The kit comprises PCR primers for quantitatively determining the amountof one or more particular species of mRNA in the pregnant woman's blood.The mRNA may encode hCG-β, hCRH, hPL, KISS1, TPFI2, or PLAC1. The kitfurther comprises a standard control that represents the amount of mRNAencoding the same protein(s) in an average woman who is healthy and notpregnant. A kit of the present invention may, in addition to or in placeof the PCR primes, comprise one or more probes that can be used toquantitatively determine the amount of mRNA encoding hCG-β, hCRH, hPL,KISS1, TPFI2, or PLAC1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates clearance of CRH mRNA from maternal plasma afterdelivery: A, CRH mRNA and B, GAPDH mRNA concentrations from maternalplasma before delivery and at 2 hours after delivery. Each linerepresents one plasma sample obtained from one subject.

FIG. 2 is a box plot of CRH mRNA concentration in maternal plasma ofpreeclamptic and control groups. CRH mRNA concentrations are expressedin copies/mL. The lines inside the boxes denote the medians. The boxesmark the interval between the 25^(th) and 75^(th) percentiles. Thewhiskers denote the interval between the 10^(th) and 90^(th)percentiles. The filled circles mark the data points outside the 10^(th)and 90^(th) percentiles.

FIG. 3 shows the levels of placenta-derived mRNA in maternal plasma innormal pregnancy. (A) Box plot of hPL mRNA in maternal plasma andprotein levels in maternal serum at different stages of gestation. (B)Box plot of βhCG mRNA in maternal plasma and hCG protein levels inmaternal serum at different stages of gestation. The lines inside theboxes denote the medians. The boxes mark the interval between the25^(th) and 75^(th) percentiles. The whiskers denote the intervalbetween the 10^(th) and 90^(th) percentiles. The filled circles mark thedata points outside the 10^(th) and 90^(th) percentiles. (C) Correlationbetween hPL mRNA in maternal plasma and protein levels in maternalserum. (D) Correlation between βhCG mRNA in maternal plasma and proteinlevels in maternal serum.

FIG. 4 shows the clearance of hPL mRNA from maternal plasma afterdelivery. (A) Maternal plasma hPL mRNA levels and (B) Maternal plasmaGAPDH mRNA levels before delivery and at 24 hours after delivery. Theresults of 8 plasma samples are shown and each line represents oneplasma sample obtained from one subject. (C) Maternal plasma hPL mRNAlevels and (D) Maternal plasma GAPDH mRNA levels before delivery and at2 hours after delivery. The results of 13 plasma samples are shown andeach line represents one plasma sample obtained from one subject.

FIG. 5 illustrates the amount of GAPDH mRNA in maternal plasma. Samples1–12 are from normal pregnant women and 28–37 are from preeclampticwomen.

FIG. 6 depicts maternal serum hCGβ mRNA concentrations infirst-trimester aneuploid and control pregnancies. Box plot of hCGβ mRNAconcentrations (common logarithmic scale) in sera of control, trisomy 21(T21) and trisomy 18 (T18) pregnancies. The lines inside the boxesdenote the medians. The boxes mark the interval between the 25^(th) and75^(th) percentiles. The whiskers denote the interval between the10^(th) and 90^(th) percentiles. The filled circles mark the data pointsoutside the 10^(th) and 90^(th) percentiles.

FIG. 7 is an outline of the strategy used for the systematicidentification of pregnancy-specific placental-expressed mRNA markers inmaternal plasma. Paired placental tissues and maternal whole bloodsamples are collected and subjected to oligonucleotide microarrayanalysis. Transcripts with increased expression in the placental tissuesrelative to whole blood are selected and their detectability in maternalplasma and pregnancy-specificity are evaluated by quantitative reversetranscriptase-PCR (QRT-PCR) on maternal plasma.

FIG. 8 depicts the clearance of placental mRNA from maternal plasmaafter delivery. Concentrations of (A) TFPI2 mRNA, (B) KISS1 mRNA, and(C) PLAC1 mRNA in maternal plasma before delivery and at 24 hours afterdelivery were measured by QRT-PCR. Each line represents one plasmasample obtained from one subject.

FIG. 9 shows correlation of normalized placental mRNA levels in maternalplasma and placental tissues. (A) Correlation of normalized placentalmRNA levels between first-trimester plasma and CVS. (B) Correlation ofnormalized placental mRNA levels between third-trimester plasma and termplacentas. The normalized levels of βhCG (nβhCG), TFPI2 (nTFPI2), KISS1(nKISS1), CRH (nCRH), and PLAC1 (nPLAC1) mRNA in plasma and placentaltissues were determined by QRT-PCR and indicated by the respectivesymbols indicated in the legends. The normalized levels for thenon-placental expressed transcripts, GAPDH (nGAPDH) and β-globin(nβ-globin) are also indicated.

DEFINITIONS

The term “preeclampsia” as used herein refers to a condition that occursduring pregnancy, the main symptom of which is various forms of highblood pressure often accompanied by the presence of proteins in theurine and edema (swelling). Preeclampsia, sometimes called toxemia ofpregnancy, is related to a more serious disorder called “eclampsia,”which is preeclampsia together with seizure. These conditions usuallydevelop during the second half of pregnancy (after 20 weeks), thoughthey may develop shortly after birth or before 20 weeks of pregnancy.

The term “chromosomal aneuploidy” as used herein refers to a state ofchromosomal abnormality where the number of chromosomes is not an exactmultiple of the usual haploid number: frequently, there is either anadditional chromosome or one missing. The most common case of achromosomal aneuploidy is a trisomy, where a single additionalchromosome is present. For example, trisomy 18 is a chromosomalabnormality where a third chromosome 18 is found in a cell, whereas athird chromosome 21 is present in the cells of a patient suffering fromtrisomy 21.

In contrast to aneuploidy, “chromosomally normal” describes the statewhere the number of chromosomes is an exact multiple of the haploidnumber, such as twice the number of chromosomes found in a haploid, andeach chromosome is present in the same number (except the sexchromosomes in the case of, e.g., male humans, where two different sexchromosomes, X and Y, are present at one copy each).

The term “pre-term labor” or “premature labor” as used herein refers tothe condition where labor that begins more than three weeks before thefull gestation period of about 40 weeks, which often leads to prematurebirth if not treated. In contrast, a pregnant woman who “delivers orwill deliver at term” as used in this application refers to one thatcarries the fetus to its full term without ever developing pre-termlabor.

The term “blood” as used herein refers to a blood sample or preparationfrom a pregnant woman or a woman being tested for possible pregnancy.The term encompasses whole blood or any fractions of blood thatessentially contain no hematopoietic or any other types of cells ofmaternal or fetal origin, including platelets. “Blood” generally doesnot contain particular matters that may contain RNA of maternal or fetalorigin. Examples of “blood” include plasma and serum. A blood samplethat is essentially free of cells is also referred to as “acellular,”where generally no platelets are present.

The term “average,” as used in the context of describing a pregnantwoman who is non-preeclamptic, carries a chromosomally normal fetus, ordoes not and will not develop pre-term labor, refers to certaincharacteristics, such as the level of mRNA encoding one or moreparticular fetal proteins found in maternal blood, that isrepresentative of a randomly selected group of women who arenon-preeclamptic, carry chromosomally normal fetuses, or do not and willnot develop pre-term labor. This selected group should comprise asufficient number of women such that the average level of mRNA encodinga particular fetal protein among these women reflects, with reasonableaccuracy, the level of mRNA in the general population of healthypregnant women with healthy fetuses. In addition, the selected group ofwomen should have a similar gestational age to that of a woman whoseblood is tested for indication of preeclampsia, fetal chromosomalaneuploidy, or pre-term labor. The preferred gestational age forpracticing the present invention may vary depends on the disorder thatis being screened for. For example, a pregnant woman is screened for therisk of preeclampsia preferably during the second trimester of thepregnancy, whereas fetal chromosomal aneuploidy is preferably screenedfor and diagnosed as early as possible. Moreover, the preferredgestational age for testing may also depend on the mRNA marker used intesting. For example, hPL mRNA is detectable throughout all threetrimesters of pregnancy, whereas hCRH mRNA becomes increasinglydetectable as gestation progresses.

The term “average” may be used similarly to refer to the amount ofspecified mRNA species that is representative of the amount found in theblood of a randomly selected group of healthy non-pregnant women.

Human chorionic gonadotropin β subunit (hCG-β), human placental lactogen(hPL), human corticotropin releasing hormone (hCRH), KiSS-1metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2(TPFI2), placenta-specific 1 (PLAC1), and glyceraldehyde-3-phosphatedehydrogenase (GAPDH), as used herein, refer to the genes (includingtheir variants and mutants) and their polynucleotide transcripts asexemplified by the sequences set forth in GenBank Accesion Nos.NM_(—)000737.2, NM_(—)022640, NM_(—)000756, U43527, NM_(—)006528,BC022335, and BC014085, respectively. In some context, these terms mayalso refer to the proteins encoded by these genes.

“Standard control” as used herein refers to a sample suitable for theuse of a method of the present invention, in order for quantitativelydetermining the amount of mRNA encoding a particular protein, e.g.,hCG-β, hCRH, hPL, KISS1, TPFI2, PLAC1, or GAPDH. Such sample contains aknown amount of the mRNA encoding a particular protein that closelyreflects the average level of such mRNA in an average pregnant woman,who is non-preeclamptic, carries a chromosomally normal fetus, or doesnot and will not develop pre-term labor, as described above. Similarly,a “standard control” may be derived from an average healthy non-pregnantwoman.

“An increase or a decrease in the amount of mRNA from the standardcontrol” as used herein refers to a positive or negative change inamount from the standard control. An increase is preferably at least2-fold, more preferably at least 5-fold, and most preferably at least10-fold. Similarly, a decrease is preferably at least 50%, morepreferably at least 80%, and most preferably at least 90%.

A “polynucleotide hybridization method” as used herein refers to amethod for detecting the presence and/or quantity of a polynucleotidebased on its ability to form Watson-Crick base-pairing, underappropriate hybridization conditions, with a polynucleotide probe of aknown sequence. Examples of such hybridization methods include Southernblotting and Northern blotting.

“PCR primers” as used herein refer to oligonucleotides that can be usedin a polymerase chain reaction (PCR) to amplify a nucleotide sequenceoriginated from an mRNA encoding a protein of interest, such as hCG-β,hCRH, hPL, KISS1, TPFI2, PLAC1, or GAPDH. At least one of the PCRprimers for amplification of a nucleotide sequence encoding anabove-named protein should be sequence-specific for the protein.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides, for the first time, methods and kits fordiagnosing, monitoring, or predicting preeclampsia, fetal chromosomalaneuploidy (such as trisomy 18 and trisomy 21), and pre-term labor inpregnant women, as well as for detecting pregnancy in women, byanalyzing the level of one or more of several mRNA species, i.e., thoseencoding, e.g., hCG-β, hCRH, hPL, KISS1, TPFI2, PLAC1, or GAPDH, presentin the women's blood.

According to the invention, the amount of mRNA encoding hCG-β, hCRH,hPL, KISS1, TPFI2, PLAC1, or GAPDH in a maternal blood sample can bequantitatively determined, preferably following an amplificationprocedure, e.g., reverse transcriptase polymerase chain reaction(RT-PCR). The amount of one of more of the above-named mRNA species isthen compared to a standard control having an mRNA level of the samespecies that is representative of an average pregnant woman withoutthese pregnancy-related disorders at a similar gestational age. Anincrease or decrease in the mRNA level indicates the presence of or anincreased risk of developing the disorders. The present invention thusprovides a novel approach for diagnosis of preeclampsia, fetalchromosomal aneuploidy, and pre-term labor, which is non-invasive aswell as gender- and polymorphism-independent.

Relying on the same methodology, by comparing the level of one or moreof the mRNA species encoding hCG-β, hCRH, hPL, KISS1, TPFI2, or PLAC1 ina woman's blood to an established control value obtained from averagenon-pregnant woman, the present invention may be used to detectpregnancy.

II. Preparation of Blood Samples

A. Obtaining Blood Samples

The first step of practicing the present invention is to obtain a bloodsample from a pregnant woman at a gestational age suitable for testingusing a method of the present invention, or from a woman who is beingtested for possible pregnancy. The suitable gestational age may varydepending on the disorder tested and sometimes the mRNA marker used, asdiscussed above. Collection of blood from a woman is performed inaccordance with the standard protocol hospitals or clinics generallyfollow. An appropriate amount of peripheral blood, e.g., between 5–20ml, is collected and maybe stored according to standard procedure priorto further preparation.

B. Preparing Acellular Blood Samples

The serum or plasma of a woman's blood is suitable for the presentinvention and can be obtained by well known methods. For example, awoman's blood can be placed in a tube containing EDTA or a specializedcommercial product such as Vacutainer SST (Becton Dickinson, FranklinLakes, N.J.) to prevent blood clotting, and plasma can then be obtainedfrom whole blood through centrifugation. On the other hand, serum isobtained through centrifugation following blood clotting. Centrifugationis typically conducted at an appropriate speed, e.g., 1,500–3,000×g, ina chilled environment, e.g., at a temperature of about 4–10° C. Plasmaor serum may be subject to additional centrifugation steps before beingtransferred to a fresh tube for RNA extraction.

III. Quantitative Determination of the Amount of mRNA in a Woman's Blood

A. Extraction of mRNA

There are numerous methods for extracting mRNA from a biological sample.The general methods of mRNA preparation (e.g., described by Sambrook andRussell, Molecular Cloning: A Laboratory Manual 3d ed., 2001) can befollowed; various commercially available reagents or kits, such asTrizol reagent (Invitrogen, Carlsbad, Calif.), Oligotex Direct mRNA Kits(Qiagen, Valencia, Calif.), RNeasy Mini Kits (Qiagen, Hilden, Germany),and PolyATtract® Series 9600™ (Promega, Madison, Wis.), may also be usedto obtain mRNA from a blood sample from a woman. Combinations of morethan one of these methods may also be used.

It is essential that all contaminating DNA be eliminated from the RNApreparations. Thus, careful handling of the samples, thorough treatmentwith DNase, and proper negative controls in the amplification andquantification steps should be used.

B. PCR-Based Quantitative Determination of mRNA Level

Once mRNA is extracted from a woman's blood sample, the amount of mRNAencoding a protein of interest, e.g., hCG-β, hCRH, hPL, KISS1, TPFI2,PLAC1, or GAPDH, may be quantified. The preferred method for determiningthe mRNA level is an amplification-based method, e.g., by PCR.

Prior to the amplification step, a DNA copy (cDNA) of the mRNA ofinterest must be synthesized. This is achieved by reverse transcription,which can be carried out as a separate step, or in a homogeneous reversetranscription-polymerase chain reaction (RT-PCR), a modification of thepolymerase chain reaction for amplifying RNA. Methods suitable for PCRamplification of ribonucleic acids are described by Romero and Rotbartin Diagnostic Molecular Biology: Principles and Applications pp.401–406;Persing et al., eds., Mayo Foundation, Rochester, Minn., 1993; Egger etal., J. Clin. Microbiol. 33:1442–1447, 1995; and U.S. Pat. No.5,075,212.

The general methods of PCR are well known in the art and are thus notdescribed in detail herein. For a review of PCR methods, protocols, andprinciples in designing primers, see, e.g., Innis, et al., PCRProtocols: A Guide to Methods and Applications, Academic Press, Inc.N.Y., 1990. PCR reagents and protocols are also available fromcommercial vendors, such as Roche Molecular Systems.

PCR is most usually carried out as an automated process with athermostable enzyme. In this process, the temperature of the reactionmixture is cycled through a denaturing region, a primer annealingregion, and an extension reaction region automatically. Machinesspecifically adapted for this purpose are commercially available.

Although PCR amplification of the target mRNA is typically used inpracticing the present invention. One of skill in the art willrecognize, however, that amplification of these mRNA species in amaternal blood sample may be accomplished by any known method, such asligase chain reaction (LCR), transcription-mediated amplification, andself-sustained sequence replication or nucleic acid sequence-basedamplification (NASBA), each of which provides sufficient amplification.More recently developed branched-DNA technology may also be used toquantitatively determining the amount of mRNA markers in maternal blood.For a review of branched-DNA signal amplification for directquantitation of nucleic acid sequences in clinical samples, see Nolte,Adv. Clin. Chem. 33:201–235, 1998.

C. Other Quantitative Methods

The mRNA of interest can also be detected using other standardtechniques, well known to those of skill in the art. Although thedetection step is typically preceded by an amplification step,amplification is not required in the methods of the invention. Forinstance, the mRNA may be identified by size fractionation (e.g., gelelectrophoresis), whether or not proceeded by an amplification step.After running a sample in an agarose or polyacrylamide gel and labelingwith ethidium bromide according to well known techniques (see, Sambrookand Russell, supra), the presence of a band of the same size as thestandard control is an indication of the presence of a target mRNA, theamount of which may then be compared to the control based on theintensity of the band. Alternatively, oligonucleotide probes specific tomRNA encoding, e.g., hCG-β, hCRH, hPL, KISS1, TPFI2, PLAC1, or GAPDH,can be used to detect the presence of such mRNA species and indicate theamount of mRNA in comparison to the standard control, based on theintensity of signal imparted by the probe.

Sequence-specific probe hybridization is a well known method ofdetecting a particular nucleic acid comprising other species of nucleicacids. Under sufficiently stringent hybridization conditions, the probeshybridize specifically only to substantially complementary sequences.The stringency of the hybridization conditions can be relaxed totolerate varying amounts of sequence mismatch.

A number of hybridization formats well known in the art, including butnot limited to, solution phase, solid phase, or mixed phasehybridization assays. The following articles provide an overview of thevarious hybridization assay formats: Singer et al., Biotechniques 4:230,1986; Haase et al., Methods in Virology, pp. 189–226, 1984; Wilkinson,In situ Hybridization, Wilkinson ed., IRL Press, Oxford UniversityPress, Oxford; and Hames and Higgins eds., Nucleic Acid Hybridization: APractical Approach, IRL Press, 1987.

The hybridization complexes are detected according to well knowntechniques and the detection is not a critical aspect of the presentinvention. Nucleic acid probes capable of specifically hybridizing to atarget nucleic acid, i.e., the mRNA or the amplified DNA, can be labeledby any one of several methods typically used to detect the presence ofhybridized nucleic acids. One common method of detection is the use ofautoradiography using probes labeled with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P, orthe like. The choice of radioactive isotope depends on researchpreferences due to ease of synthesis, stability, and half lives of theselected isotopes. Other labels include compounds (e.g., biotin anddigoxigenin), which bind to antiligands or antibodies labeled withfluorophores, chemiluminescent agents, and enzymes. Alternatively,probes can be conjugated directly with labels such as fluorophores,chemiluminescent agents or enzymes. The choice of label depends onsensitivity required, ease of conjugation with the probe, stabilityrequirements, and available instrumentation.

The probes and primers necessary for practicing the present inventioncan be synthesized and labeled using well known techniques.Oligonucleotides used as probes and primers may be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage and Caruthers, Tetrahedron Letts.,22:1859–1862, 1981, using an automated synthesizer, as described inNeedham-VanDevanter et al., Nucleic Acids Res. 12:6159–6168, 1984.Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson andRegnier, J. Chrom., 255:137–149, 1983.

IV. Establishing a Standard Control

In order to establish a standard control, a group of healthy pregnantwomen carrying healthy fetuses should first be selected. These womenshould be of similar gestational age, which is within the appropriatetime period of pregnancy for screening of conditions such aspreeclampsia, fetal chromosomal aneuploidy, and pre-term labor using themethods of the present invention. Similarly, a standard control isestablished using samples from a group of healthy non-pregnant women.

The healthy status of the selected pregnant women and the fetuses theyare carrying should be confirmed by well established, routinely employedmethods including but not limited to monitoring blood pressure of thewomen, recording the onset of labor, and conducting fetal geneticanalysis using CVS and amniocentesis.

Furthermore, the selected group of healthy pregnant women carryinghealthy fetuses or healthy non-pregnant women must be of a reasonablesize, such that the average amount of mRNA encoding hCG-β, hCRH, hPL,KISS1, TPFI2, PLAC1, or GAPDH calculated from the group can bereasonably regarded as representative of the normal or average amountamong the general population of healthy women carrying healthy fetusesor healthy non-pregnant women. Preferably, the selected group comprisesat least 10 women.

Once an average value is established for the amount of mRNA encoding anyone protein based on the individual values found in each women of theselected group, this values is considered a standard for the mRNAspecies. Any blood sample that contains a similar amount of mRNA of thesame species can thus be used as a standard control. A solutioncontaining mRNA encoding hCG-β, hCRH, hPL, KISS1, TPFI2, PLAC1, or GAPDHwith a concentration of the established average of the same species canalso be artificially assembled and serve as a standard control.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

EXAMPLES Example 1 Elevated hCRH mRNA Level in Preeclamptic Women

A. Methods

Subjects

Peripheral blood samples were collected with informed consent andResearch Ethics Committee approval from pregnant women, who attended theDepartment of Obstetrics and Gynecology at the Prince of Wales Hospital,Hong Kong.

In the first part of this study, blood samples were obtained from 10healthy pregnant women during the third trimester of gestation. In thesecond part of the project, 4 pregnant women with uncomplicatedpregnancy were recruited just prior to elective cesarean section.Peripheral blood samples were taken from these subjects just prior todelivery and at 2 hours post-delivery. In the third part of the study,two patient groups were studied: (a) 12 preeclamptic women and (b) 10control pregnancies. The median gestational ages of the preeclamptic andcontrol groups were 37 weeks and 38 weeks, respectively. Preeclampsiawas defined on the basis of a sustained increase in diastolic bloodpressure >110 mm Hg on one occasion or >90 mm Hg on two or moreoccasions at least 4 hours apart, with the presence of significantproteinuria in women with no history of hypertension. Significantproteinuria was defined as proteinuria >0.3 g/day or ≧9+ on dipsticktesting in two clean-catch midstream urine specimens collected at least4 hours apart. The control group included pregnant women with nopreexisting medical diseases or antenatal complications.

Processing of Blood Samples

Blood samples were processed based on a previously reported protocol (Nget al., Clin. Chem. 48:1212–1217, 2002). In brief, 10-mL blood sampleswere collected in EDTA-containing tubes, and centrifuged at 1600×g for10 min at 4° C. Plasma was then carefully transferred into plainpolypropylene tubes. The plasma samples were re-centrifuged at 16000×gfor 10 min at 4° C., and the supernatants were collected into freshpolypropylene tubes.

RNA Extraction

1.6 mL of plasma was mixed with 2 mL of Trizol LS reagent (Invitrogen,Carlsbad, Calif.) and 0.4 mL of chloroform, as describe by Ng et al.,supra. The mixture was centrifuged at 11,900×g for 15 min at 4° C. andthe aqueous layer was transferred into new tubes. One volume of 70%ethanol was added to one volume of the aqueous layer. The mixture wasthen applied to an RNeasy mini column (Qiagen, Hilden, Germany) and wasprocessed according to the manufacturer's recommendations. Total RNA waseluted with 30 μL of RNase-free water and stored at −80° C. DNasetreatment (RNase-Free DNase Set, Qiagen, Hilden, Germany) was carriedout to remove any contaminating DNA.

Real-Time Quantitative RT-PCR

One-step real-time quantitative RT-PCR was used for all mRNAquantitation according to the protocol provided by Ng et al., supra. TheCRH primer sequences were 5′-GCCTCCCATCTCCCTGGAT-3′ (forward; SEQ IDNO:1) and 5′-TGTGAGCTTGCTGTGCTAACTG-3′ (reverse; SEQ ID NO:2), and thedual-labelled fluorescent probe was5′-(FAM)TCCTCCGGGAAGTCTTGGAAATGGC(TAMRA)-3′ (SEQ ID NO:3). Calibrationcurves for CRH mRNA quantifications were prepared by serial dilutions ofhigh performance liquid chromatography-purified single strandedsynthetic DNA oligonucleotides (Genset Oligos, Singapore) specifying a89 bp CRH amplicon (Genbank Accession No. NM_(—)000756), withconcentrations ranging from 1×10⁷ copies to 1×10¹ copies. Absoluteconcentrations of CRH mRNA were expressed as copies/mL of plasma. Thesequences of the synthetic DNA oligonucleotides for CRH calibrationswere 5′-GGAGCCTCCCATCTCCCTGGATCTCACCTTCCACCTCCTCCGGGAAGTCTTGGAAATGGCCAGGGCCGAGCAGTTAGCACAGCAAGCTCACAGCA-3′ (SEQ ID NO:4). A calibrationcurve for GAPDH quantification was prepared as previously described,with results expressed in pg/mL plasma (Ng et al., supra).

The RT-PCR reactions were set up according to the manufacturer'sinstructions (EZ rTth RNA PCR reagent set, Applied Biosystems, FosterCity, Calif.) in a reaction volume of 25 μL. The fluorescent probes(Genset Oligos) were used at concentrations of 100 nM. The PCR primers(Genset Oligos) were used at a concentration of 200 nM for both the CRHand GAPDH systems. 5 μL of extracted plasma RNA was used foramplification. Each sample was analyzed in duplicate, and thecorresponding calibration curve was run in parallel with each analysis.Samples were also tested to ensure they were negative for DNA bysubstituting the rTth polymerase with the AmpliTaq Gold enzyme (AppliedBiosystems, Foster City, Calif.). No amplification was observed for thiscontrol analysis, indicating the specificity of the assays for therespective mRNA. Multiple negative water blanks were also included inevery analysis.

The thermal profile used for the CRH and GAPDH analysis was as follows:the reaction was initiated at 50° C. for 2 min for the included uracilN-glycosylase to act, followed by reverse transcription at 60° C. for 30min. After a 5-min denaturation at 95° C., 40 cycles of PCR was carriedout using denaturation at 94° C. for 20 s and 1 min annealing/extensionat 58° C. and 62° C. for the CRH and GAPDH systems, respectively.

Statistical Analysis

Statistical analysis was performed using the Sigma Stat 2.03 software(SPSS).

B. Results

Establishment of Real-Time Quantitative RT-PCR

To determine the quantitative performance of the CRH RT-PCR assay, weused this system to amplify serially diluted calibrators which weresynthetic DNA oligonucleotides based on the CRH sequence. Previous datahave shown that such single stranded oligonucleotides reliably mimic theproducts of the reverse transcription step and produce calibrationcurves that are identical to those obtained using T7-transcribed RNA(Bustin, J. Mol. Endocrinol. 25:169–193, 2000). The calibration curvefor the CRH amplification systems demonstrated a dynamic range from2.5×10¹ to 1×10⁶ copies and had a correlation coefficient of 0.983. Thesensitivities of the amplification steps of these assays were sufficientto detect 25 copies of the CRH target. To determine the precision of thewhole analytical procedure involving RNA extraction, reversetranscription and amplification steps, we performed 10 replicate RNAextractions from a plasma sample obtained from a healthy pregnant woman(gestational age: 38 weeks) and subjected these extracted RNA samples toRT-PCR analysis. The coefficient of variation of the Ct values of thesereplicate analyses for CRH mRNA was 2.8%. The development andperformance of the real-time quantitative GAPDH RT-PCR assay waspreviously described by Ng et al., supra.

Detectability of CRH mRNA in Maternal Plasma

To test whether CRH mRNA transcripts were detectable in maternal plasma,plasma samples from 10 pregnant women at the third trimester ofpregnancy (gestational age 37 to 41 weeks) were analyzed by the CRHRT-PCR assay. CRH mRNA was detected in all tested samples. The medianconcentration of plasma CRH mRNA was 73 copies/mL (interquartile range:51 to 177). As a positive control, GAPDH mRNA was detectable in all ofthese plasma samples.

Clearance of CRH mRNA From Maternal Plasma Following Delivery

To demonstrate that the maternal plasma CRH mRNA was derived from thefeto-placental unit, we analyzed plasma from 4 women for CRH mRNA bothbefore and at 2 hours post-delivery. Thus, CRH mRNA was detected in all4 pre-delivery maternal plasma samples. In contrast, CRH mRNA was notdetected in any of the post-delivery samples. GAPDH mRNA was detectablein all plasma samples, thus demonstrating the quality of the samples.The data are shown in FIGS. 1A and B.

Quantitative Analysis of CRH mRNA in the Plasma of Preeclamptic PregnantWomen

To compare the concentration of CRH mRNA in maternal plasma ofpreeclamptic and control pregnant women, plasma samples from 12preeclamptic women and 10 control pregnant women with matchedgestational age were obtained. FIG. 2 shows that the median CRH mRNAconcentration in the plasma of preeclamptic women and controlpregnancies were 1070 copies/mL (interquartile range, 535 to 1468) and102 copies/mL (interquartile range, 51 to 158), respectively. The medianplasma CRH mRNA concentrations were 10.5 times higher in preeclampticthan control pregnancies (Mann-Whitney test, P<0.001).

C. Conclusion

Plasma CRH mRNA represents a new molecular marker for preeclampsia.Compared with maternal plasma fetal DNA analysis, plasma RNA analysishas the advantage that it is gender- and polymorphism-independent. It isenvisioned that plasma RNA analysis may ultimately allow non-invasivegene expression profiling of an unborn fetus.

Example 2 Detection of hPL and hCG-β mRNA in the Plasma of PregnantWomen

A. Methods

Subjects

15-ml blood samples were collected with informed consent and ResearchEthics Committee approval from healthy women with singletonuncomplicated pregnancies, who attended the Department of Obstetrics andGynecology at the Prince of Wales Hospital, Hong Kong.

Processing of Blood Samples

The blood samples were collected in EDTA-containing and plain tubes, andcentrifuged at 1600×g for 10 min at 4° C. Plasma and serum were thencarefully transferred into plain polypropylene tubes. The serum sampleswere stored at −20° C. for immunoassays for the hPL and hCG-β proteins.The plasma samples were re-centriftiged at 16000×g for 10 min at 4° C.,and the supernatants were collected into fresh polypropylene tubes. Allplacental tissue samples were immediately stored in an RNA Laterstabilizing solution (Ambion, Austin, Tex.) and kept at −80° C. untilRNA extraction. For the filtration study, plasma samples were dividedinto three portions: two were individually passed through filters(Millex-GV; Millipore China Limited, Hong Kong) with pore sizes ofeither 0.45 μm or 5 μm. The third portion was not subjected tofiltration.

RNA Extraction

For plasma samples, 1.6 ml of plasma was mixed with 2 ml of Trizol LSreagent (Invitrogen, Carlsbad, Calif.) and 0.4 ml of chloroformaccording to the protocol of Ng et al., supra. For placental tissues,samples were homogenized in Trizol reagent (Invitrogen) and chloroformwas then added according to the manufacturer's recommendations. Themixture was centrifuged at 11,900×g for 15 min at 4° C. and the aqueouslayer was transferred into new tubes. One volume of 70% ethanol wasadded to one volume of the aqueous layer. The mixture was then appliedto an RNeasy mini column (RNeasy Mini Kit, Qiagen, Hilden, Germany) andwas processed according to the manufacturer's recommendations. Total RNAwas eluted with 30 μl of RNase-free water and stored at −80° C. DNasetreatment was carried out to remove any contaminating DNA (RNase-FreeDNase Set, Qiagen, Hilden, Germany).

Real-Time Quantitative RT-PCR

One-step real-time quantitative RT-PCR was used for all mRNAquantitation as described by Ng et al., supra. The primers for all ofthe hPL, hCG-β and GAPDH RT-PCR assays were intron-spanning. The hPLprimer sequences were 5′-CATGACTCCCAGACCTCCTTC-3′ (sense; SEQ ID NO:5)and 5′-TGCGGAGCAGCTCTAGATTG-3′ (antisense; SEQ ID NO:6), and thedual-labeled fluorescent probe was5′-(FAM)TTCTGTTGCGTTTCCTCCATGTTGG(TAMRA)-3′ (SEQ ID NO:7). The hCG-βprimer sequences were 5′-CTACTGCCCCACCATGACCC-3′ (sense; SEQ ID NO:8)and 5′-TGGACTCGAAGCGCACATC-3′ (antisense; SEQ ID NO:9), and thedual-labeled fluorescent probe was5′-(FAM)CCTGCCTCAGGTGGTGTGCAACTAC(TAMRA)-3′ (SEQ ID NO:10). Calibrationcurves for hPL and hCG-β quantifications were prepared by serialdilutions of high performance liquid chromatography-purified singlestranded synthetic DNA oligonucleotides (Genset Oligos, Singapore)specifying the hPL and hCG-β amplicons, respectively, withconcentrations ranging from 1×10⁷ copies to 1×10¹ copies. These assayswere able to detect 100 copies of the respective calibrator targets.Absolute concentrations of hPL and hCG-β mRNA were expressed ascopies/ml of plasma. Previous data have shown that such single strandedoligonucleotides reliably mimic the products of the reversetranscription step and produce calibration curves that are identical tothose obtained using T7-transcribed RNA (Bustin, supra). The sequencesof the synthetic DNA oligonucleotides for hPL and hCG-β calibrationswere 5′-TGCGGAGCAGCTCTAGATTGGATTTCTGTTGCGTTTCCTCCATGTTGGAGGGTGTCGGAATAGAGTCTGAGAAGCAGAAGGAGGTCTGGGAGTCATGC-3′ (SEQ ID NO:11) and5′-GATGGACTCGAAGCGCACATCGCGGTAGTTGCACACCACCTGAGGCAGGGCCGGCAGGACCCCCTGCAGCACGCGGGTCATGGTGGGGCAGTAGCC-3′ (SEQ ID NO:12),respectively. A calibration curve for GAPDH quantification was preparedas previously described by Ng et al, supra, with results expressed inpg/ml plasma.

The RT-PCR reactions were set up according to the manufacturer'sinstructions (EZ rTth RNA PCR reagent set, Applied Biosystems, FosterCity, Calif.) in a reaction volume of 50 μl. The fluorescent probes(Genset Oligos) were used at concentrations of 100 nM. The PCR primers(Genset Oligos) were used at a concentration of 300 nM for hPL and 200nM for both hCG-β and GAPDH. 5 μl of extracted plasma RNA and 0.1 ng ofextracted total placental RNA were used for amplification. Each samplewas analyzed in duplicate, and the corresponding calibration curve wasrun in parallel for each analysis. Samples were also tested to ensurethey were negative for DNA by substituting the rTth polymerase with theAmpliTaq Gold enzyme (Applied Biosystems, Foster City, Calif.). Noamplification was observed for this control analysis, indicating thespecificity of the assays for the respective mRNA. Multiple negativewater blanks were also included in every analysis.

The thermal profile used for the hPL, hCG-β, and GAPDH analysis was asfollows: the reaction was initiated at 50° C. for 2 min for the includeduracil N-glycosylase to act, followed by reverse transcription at 60° C.for 30 min. After a 5-min denaturation at 95° C., 40 cycles of PCR wascarried out using denaturation at 94° C. for 20 s and 1 minannealing/extension at 56° C., 58° C. and 60° C. for hPL, hCG-β, andGAPDH, respectively.

Replicate RNA extraction and RT-PCR analysis indicated that thecoefficients of variation of Ct values of the analytical system for hPLand hCG-β mRNA were 2.3% and 3.2%, respectively.

Protein Assays

The hPL and hCG protein concentrations were determined in maternal serumby a radioimmunoassay (Diagnostic Products Corp., Los Angeles, Calif.)and an electrochemiluminescence immunoassay (Roche modular E170),respectively.

B. Results

Detectability and Stability of Placental mRNA in Maternal Blood

We obtained peripheral blood samples from 10 pregnant women (gestationalage 7 to 14 weeks). In half of the blood sample from each subject,plasma harvesting and RNA extraction were performed immediately uponarrival at the laboratory (within 1 hour of venesection). To test if hPLand hCG-β mRNA transcripts were detectable in maternal plasma, weanalyzed the extracted RNA using the respective real-time RT-PCR assays.We observed hPL and hCG-β mRNA signals in all 10 plasma samples. Theseresults demonstrated that placental mRNA was indeed detectable in theplasma of pregnant women. As a positive control, GAPDH mRNA was alsodetectable in all of these plasma samples.

To investigate the stability of placental mRNA in maternal blood, weleft the remaining aliquot from each of these maternal blood samples for24 hours at room temperature. We then extracted RNA from these samplesand measured the levels of hPL, hCG-β, and GAPDH transcripts. Nosignificant difference was observed in the levels of hPL and hCG-β mRNAtranscripts, while the GAPDH mRNA was significantly higher in samplesthat had been left at room temperature for 24 hours than those that hadbeen processed immediately (Wilcoxon test, P=0.770 for the hPL study;P=0.275 for the hCG-β study; P<0.05 for the GAPDH study). These resultsindicate that hPL and hCG-fl mRNA in plasma was stable for up to 24hours at room temperature.

Variation of Circulating Placental mRNA with Gestational Age

We obtained plasma samples from 39 pregnant women at different stages ofgestation. We detected hPL mRNA in 100% (39 out of 39) of all plasmatested in all three trimesters of pregnancy. For hCG-β mRNA, thedetection rates were 100% (14/14) for first trimester (gestational age:7 to 14 weeks) samples, 42% (5/12) for second trimester (gestationalage: 15 to 23 weeks) samples and 7.7% (1/13) for third trimester(gestational age: 35 to 41 weeks) samples. In the first-trimester plasmasamples, the median levels of hPL and hCG-β mRNA were 2671(interquartile range: 375 to 6217) and 1205 copies/ml (interquartilerange: 566 to 1927), respectively (FIGS. 3A and B). The median levels ofplasma hPL and hCG-β mRNA from second-trimester pregnancies were 4784(interquartile range: 2679 to 10139) and 0 copies/ml (interquartilerange: 0 to 125), respectively. In the third-trimester plasma samples,the median levels of plasma hPL and hCG-β mRNA were 13869 (interquartilerange: 7829 to 27434) and 0 copies/ml (interquartile range: 0 to 0),respectively. Overall, circulating hPL and hCG-f mRNA levels show anincreasing and a decreasing trend, respectively, with gestational age.The corresponding levels of hPL and hCG-β proteins were also determined(FIGS. 3A and B). The overall gestational variation of circulating hPLand hCG-β mRNA shows a resemblance to the trends exhibited by thecorresponding proteins (Pearson correlation analysis, r=0.622; P<0.001for hPL and r=0.784; P<0.001 for hCG-β, FIGS. 3C and D).

Rapid Clearance of Placental mRNA from Maternal Plasma FollowingDelivery

We next investigated if delivery would result in the clearance ofplacental mRNA from maternal plasma. We chose hPL mRNA as our targetbecause of the relative abundance of hPL mRNA in maternal plasma duringthe last trimester of pregnancy (gestational age of studied subjects: 38to 42 weeks). In 8 pre-delivery plasma samples, the median level of hPLmRNA transcripts was 50004 copies/ml. hPL mRNA was not detected in anyof the postpartum samples at 24 hours after delivery (FIG. 4A). As acontrol, GAPDH mRNA was detected in all pre- and post-delivery plasmasamples (FIG. 4B). No systematic alteration in maternal plasma GAPDHmRNA levels was observed (Wilcoxon test, P=0.313). We next investigatedif clearance of circulating hPL mRNA might be observable if a shorterpost-partum time-point (2 hours) was studied. In 13 subjects recruitedfor this second study, the median pre-delivery level of hPL mRNA was24499 copies/ml (FIG. 4C). Nine of the thirteen women had no detectableplasma placental RNA by 2 hours postpartum. The remaining subjects hadapproximately 66–97% of maternal plasma fetal RNA cleared by 2 hoursafter delivery. We detected GAPDH mRNA in all plasma samples, thusdemonstrating the quality of the samples (FIG. 4D). No systematicalteration in maternal plasma GAPDH mRNA levels was observed (Wilcoxontest, P=1.000).

Example 3 Elevation in Maternal Plasma GAPDH mRNA in Preeclamptic Women

Pregnant women attending the Department of Obstetrics and Gynecology atthe Prince of Wales Hospital were recruited with informed consent.Preeclampsia was diagnosed using the criteria as indicated in Example 1.

A. Methods

Maternal blood samples were taken into heparinized tubes and processedas indicated in Example 1. Plasma RNA was extracted and GAPDH mRNA levelwas quantified as indicated in Example 1.

B. Results

Elevation in maternal plasma GAPDH mRNA concentrations was observed inthe preeclamptic group, when compared with the control group (FIG. 5).The median maternal plasma GAPDH mRNA concentrations in control andpreeclamptic subjects were 70 pg/ml and 281 pg/ml, respectively. Thedifference is statistically significant (Mann-Whitney test, p<0.005).

C. Conclusion

Maternal plasma GAPDH mRNA is a new noninvasive marker for preeclampsia.

Example 4 hCGβ mRNA Concentrations in Maternal Serum in AneuploidPregnancies

A. Methods

The concentration of βhCG mRNA was measured by real-time quantitativeRT-PCR in first-trimester serum samples collected from 141 pregnantwomen investigated between January and August 2003. In all studiedsubjects, chorionic villous sampling (CVS) for fetal karyotyping wascarried out. All maternal blood samples were collected immediatelybefore CVS. Blood samples were collected in plain tubes, and centrifugedat 1600×g for 10 min at 4° C. Serum was then transferred into plainpolypropylene tubes. 3.2 mL serum was immediately stored in 4 mL Trizoland kept at −80° C. until RNA extraction. Serum RNA was extracted from1.6 mL of serum by a modified RNeasy RNA Mini Kit (Qiagen, Hilden,Germany) as previously described (Ng et al., Proc. Natl. Acad. Sci. USA,100:4748–4753, 2003). Total RNA was eluted with 30 μL of RNase-freewater and stored at −80° C. DNase treatment was carried out to removeany contaminating DNA (RNase-Free DNase Set, Qiagen, Hilden, Germany).

One-step real-time quantitative RT-PCR was used for βhCG mRNAquantification, as described by Ng et al., Proc. Natl. Acad. Sci. USA,100:4748–4753, 2003. The RT-PCR reactions were set up in a reactionvolume of 25 μL. The primers and fluorescent probe were used atconcentrations of 300 nM and 100 nM, respectively. 6 μL of extractedserum RNA was used for amplification. The thermal profile used for theanalysis was as follows: the reaction was initiated at 50° C. for 2 minfor the included uracil N-glycosylase to act, followed by reversetranscription at 60° C. for 30 min. After a 5-min denaturation at 95° C.40 cycles of PCR was carried out using denaturation at 94° C. for 20 sand 1 min annealing/extension at 57° C. The sensitivity, linearity, andprecision of the assay have been established by Ng et al., Proc. Natl.Acad. Sci. USA, 100:4748–4753, 2003. As little as 100 copies of thesynthetic oligonucleotide were detectable in the reaction mixture.Concentrations of serum hCGβ mRNA were expressed as copies/mL of serum.As no recovery experiments had been done, the reported concentrations(copies/mL) were minimum estimates.

Among the 149 pregnant women recruited, 15 women carried fetuses withtrisomy 21 and 11 carried fetuses with trisomy 18. The remaining of 123cases had euploid fetuses and served as controls. The median gestationalage of the controls was 12.5 weeks (range: 11.2 to 14.3 weeks). Themedian gestational ages of the trisomy 21 and trisomy 18 cases were 12.5weeks (range: 12.1 to 14.2 weeks) and 12.3 weeks (range: 11.4 to 14.1weeks), respectively. No significant difference of the gestational agewas observed amongst the three cohorts (Kruskal-Wallis, P=0.706).

B. Results

Maternal serum samples of the 149 studied cases were subjected to hCGβmRNA quantification. hCGβ mRNA could be detected in the maternal serumof 140 out of 149 pregnancies (94%). In the control cohort, thedetection rate of hCGβ mRNA was 96.7% (119 of 123). For the trisomy 21and trisomy 18 cohorts, the detection rates were 93.3% (14 of 15) and63.6% (i of 11), respectively. The median serum hCGβ mRNA concentrationsof the three cohorts were 6108 copies/mL (interquartile range: 2867 to19249 copies/mL) for the control cohort, 13165 copies/mL (interquartilerange: 4403 to 25265 copies/mL) for the trisomy 21 cohort, and 652copies/mL (interquartile range: 0 to 11662 copies/mL) for the trisomy 18cohort (FIG. 6). The difference in the median hCGβ mRNA concentrationsamong the three cohorts is statistically significant (Kruskal-Wallis,P=0.024). Pairwise multiple comparisons were performed and showed thatsignificant differences were observed between the trisomy 18 and controlcases (Dunn's test, P<0.05), and between the trisomy 18 and trisomy 21cases (Dunn's test, P<0.05). No statistically significant difference wasobserved between the serum hCGβ mRNA concentrations in trisomy 21 andcontrol cases (Dunn's test, P>0.05), due to a limited sample size. Givena larger sample size, however, a difference of statisticallysignificance will be established.

C. Conclusion

This study has confirmed that circulating hCGβ mRNA is easily androbustly detectable in the serum of first-trimester pregnant women, witha detection rate of 94%. These data demonstrate that the medianconcentration of serum hCGβ mRNA in trisomy 18 pregnancies was about 10%of the median concentration in the control pregnancies and statisticallysignificant difference was observed. On the other hand, although themedian concentration of serum hCGβ mRNA in trisomy 21 pregnancies was2.2-fold higher than the median concentration in the controlpregnancies, no statistically significant difference was observed due tolimited sample size. Such statistically significant difference isexpected in a study of larger scale. These data offer the firstdemonstration that circulating hCGβ mRNA concentration in thefirst-trimester serum of trisomy 18 pregnancies is significantlyreduced, whereas the circulating hCGβ mRNA concentration is elevated intrisomy 21 pregnancies.

These data indicate the diagnostic usefulness of circulating hCGβ mRNAas a marker for predicting aneuploid, e.g., trisomy 18 and trisomy 21,pregnancies. An overlap in the hCGβ mRNA concentrations between thetrisomy 18 and control cases was observed in this study. This impliesthat a relatively low sensitivity and specificity might result ifmaternal serum hCGβ mRNA measurement is used as the sole predictor forpregnancies with trisomy 18. Additional markers, such as hPL, hCRH,KiSS-1, TPFI2, and PLAC1, can be used in combination with hCGβ toachieve enhanced sensitivity and specificity in diagnosis.

Example 5 Identification of Placental mRNA in Maternal Plasma

A. Methods

The study was performed in two stages. Initially, placental tissue geneexpression profiles in both the first and third trimesters of pregnancywere systematically identified using oligonucleotide microarrays. Thiswas followed by the development of a number of assays based on real-timequantitative reverse-transcriptase polymerase chain reaction (QRT-PCR)for the detection of six of the identified placental-expressed genes inmaternal plasma. The RNA transcripts studied were assessed both fortheir detectability in maternal plasma, and the correlation of theirlevels between plasma and placental tissues. All placental and bloodsamples used in this study were collected with informed consent fromhealthy women with singleton uncomplicated pregnancies, who attended theDepartment of Obstetrics and Gynaecology at the Prince of WalesHospital, Hong Kong.

Identification of Placental Gene Expression Profiles

Five each of first-trimester (gestational age range: 9 to 12 weeks) andthird-trimester (gestational age range: 38 to 40 weeks) placental tissuesamples were obtained from pregnant women by chorionic villus sampling(CVS) before abortions or immediately after elective caesarean delivery,respectively. The placental tissue samples were stored in RNAlater™(Ambion®, Austin, Tex.) immediately upon collection and kept at −80° C.until RNA extraction. 6 mL of peripheral blood was collectedconcurrently at the time of tissue collection and stored in PAXgene™Blood RNA Tubes (PreAnalytiX, Hombrechtikon, Switzerland).

Total RNA from placental tissues were extracted with the Trizol Reagent(Invitrogen, Carlsbad, Calif.) and purified with an RNeasy mini-kit(Qiagen, Hilden, Germany) following manufacturer's protocols. Total RNAfrom peripheral blood was extracted by the PAXgene™ Blood RNA Kit(PreAnalytiX, Hombrechtikon, Switzerland) according to manufacturer'sinstructions, with the inclusion of DNase treatment (RNase-Free DNaseSet, Qiagen, Hilden, Germany).

For each sample, ten microgrammes of the extracted RNA were labeled andhybridized to the GeneChip® Human Genome U133A Arrays (Affymetrix, SantaClara, Calif.) according to the manufacturer's instructions. Afterhybridization, each array was washed and stained in a GeneChip® FluidicsStation 400 (Affymetrix, Santa Clara, Calif.). The chips were scannedwith the GeneArray Scanner (Affymetrix, Santa Clara, Calif.) andanalyzed using the GeneChip® Microarray Suite 5.0 (Affymetrix).

Quantitative Assessment of Placental-expressed RNA Transcripts inMaternal Plasma by Real-time QRT-PCR

Paired placentas and maternal whole blood samples from 10 first and 10third trimester pregnancies were collected. Peripheral blood samplesfrom 10 pregnant women before and after delivery were also recruited.The placental tissues were processed as described above. 12 mL of theblood samples was collected into EDTA tubes and was centrifuged at1600×g for 10 min at 4° C. Plasma was then carefully transferred intoplain polypropylene tubes. The plasma samples were re-centrifuged at16000×g for 10 min at 4° C. Supernatants were collected into freshpolypropylene tubes. RNA extraction from maternal plasma was performedas previously described by Ng. et al., Clin. Chem., 48:1212-1217, 2002.DNase treatment (RNase-Free DNase Set, Qiagen, Hilden, Germany) wascarried out to remove any contaminating DNA.

The study focused on the assessment of six placental-expressed mRNAtranscripts that were identified from the placental microarray geneexpression profiles, including human placental lactogen (hPL), humanchorionic gonadotropin beta subunit (βhCG), corticotropin releasinghormone (CRH), tissue factor pathway inhibitor 2 (TFPI2), KiSS-1metastasis-suppressor (KISS1) and placenta-specific 1 (PLAC1).Quantitative analysis of two non-placental-specific transcripts,glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-haemoglobin(β-globin) mRNA was performed as controls.

QRT-PCR assays for the detection of GAPDH, hPL, hCGβ, and CRH mRNA weredescribed previously (Ng. et al., Clin. Chem., 48:1212–1217, 2002; Ng etal., Proc. Natl. Acad. Sci. USA, 100:4748–4753, 2003; and Ng et al.,Clin. Chem., 49:727–731, 2003). The primer sequences for the TFPI2 assaywere 5′-ACAAATTTCTACACCTGGGAGGC-3′ (sense; SEQ ID NO:13) and5′-CGGCAAACTTTGGGAACTTTT-3′ (antisense; SEQ ID NO:14), and thedual-labeled fluorescent probe was 5′-(FAM) TGCGACGATGCTTGCTGGAGGA(TAMRA)-3′ (SEQ ID NO:15). FAM and TAMRA represented6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively.The primer sequences for KISS1 quantification were5′-GCCCAGGCCAGGACTGA-3′ (sense; SEQ ID NO:16) and5′-GCCAAGAAACCAGTGAGTTCATC-3′ (antisense; SEQ ID NO:17), and thedual-labelled fluorescent probe was 5′-(FAM)CCTCAAGGCACTTCTAGGACCTGGCTCTTC (TAMRA)-3′ (SEQ ID NO:18). The PLAC1assay primer sequences were 5′-ATTATCCCCAGCTGCCAGAA-3′ (sense; SEQ IDNO:19) and 5′-GCAGCCAATCAGATAATGAACCA-3′ (antisense; SEQ ID NO:20), andthe dual-labelled fluorescent probe was 5′-(FAM)AAGAAATCCTCACTGGACGGCTTCCTG (TAMRA)-3′ (SEQ ID NO:21). The primersequences for the β-globin assay were 5′-GCTGCACTGTGACAAGCTGC-3′ (sense;SEQ ID NO:22) and 5′ GCACACAGACCAGCACGTTG-3′ (antisense; SEQ ID NO:23),and the fluorescent probe was 5′-(FAM) CGTGGATCCTGAGAACTTCAGGCTC(TAMRA)-3′ (SEQ ID NO:24).

Calibration curves for hPL, βhCG, CRH, TFPI2, KISS1, PLAC1, and β-globinmRNA quantification were prepared by serial dilutions of highperformance liquid chromatography-purified single stranded synthetic DNAoligonucleotides as described by Bustin et al., J. Mol. Endocrinol.,25:169–193, 2000 (Genset Oligos, Singapore) specific for the respectiveamplicons, with concentrations ranging from 1×10⁶ copies to 10 copies.The sequences of the synthetic DNA oligonucleotides for hPL, βhCG andCRH calibration were described previously (see Ng et al., Proc. Natl.Acad. Sci. USA, 100:4748–4753, 2003 and Ng et al., Clin. Chem.,49:727–731, 2003). The sequences of the synthetic DNA oligonucleotidesfor TFPI2, KISS1, PLAC1, and β-globin calibrators were5′-CGCCAACAATTTCTACACCTGGGAGGCTTGCGACGATGCTTGCTGGAGGATAGAAAAAGTTCCCAAAGTTTGCCGGCTG-3′ (SEQ ID NO:25),5′-CTGCCCAGGCCAGGACTGAGGCAAGCCTCAAGGCACTTCTAGGACCTGGCTCTTCTCACCAAGATGAACTCACTGGTTTCTTGGCAG-3′ (SEQ ID NO:26),5′-ACAAATTATCCCCAGCTGCCAGAAGAAGAAATCCTCACTGGACGGCTTCCTGTTTCCTGTGGTTCATTATCTGATTGGCTGCAGG-3′ (SEQ ID NO:27) and5-TGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGG-3′ (SEQ ID NO:28) respectively. Except for GAPDHmRNA, absolute concentrations of all transcripts were expressed ascopies/ng of total placental RNA and copies/ml of plasma for placentaltissues and maternal plasma, respectively. The calibration curve forGAPDH quantification was prepared by serial dilutions of human total RNA(Ng. et al., Clin. Chem., 48:1212–1217, 2002).

The QRT-PCR reactions were set up according to the manufacturer'sinstructions (EZ rtth RNA PCR reagent set, Applied Biosystems, FosterCity, Calif., USA) in a reaction volume of 50 μl. The QRT-PCR assayswere carried out in a combined thermal cycler and fluorescent detector(ABI Prism 7700, Applied Biosystems, Foster City, Calif., USA). Thereaction conditions for the GAPDH, hPL, βhCG and CRH QRT-PCR assays weredescribed previously (Ng. et al., Clin. Chem., 48:1212–1217, 2002; Ng etal., Proc. Natl. Acad. Sci. USA, 100:4748–4753, 2003; and Ng et al.,Clin. Chem., 49:727–731, 2003). For the other three transcripts, the PCRprimers (Geneset Oligos, Singapore) were used at concentrations of 200nM for TFPI2 and β-globin, 300 nM for KISS1 and 400 nM for PLAC1. Thefluorescent probes (Applied Biosystems, Foster City, Calif., USA) wereused at concentrations of 80 nM for TFPI2, 150 nM for KISS1, 200 nM forPLAC1 and 300 nM for β-globin. Before performing QRT-PCR, contaminatingDNA in the extracted placental tissue RNA was removed by DNase Idigestion (Invitrogen, Carlsbad, Calif.) according to manufacturer'srecommendations. 0.4 ng of extracted placental RNA and 6 μl of extractedplasma RNA were used for amplification. Multiple negative water blankswere included in every analysis.

The thermal profiles used for TFPI2, KISS1, PLAC1, and β-globin mRNAanalysis were as follows: the reaction was initiated at 50° C. for 2 minfor the included uracil N-glycosylase to act, followed by reversetranscription at 60° C. for 30 min. After a 5-min denaturation at 95°C., 40 cycles of PCR were carried out using denaturation at 92° C. for15 s and 1 min annealing/extension at 56° C. for PLAC1, 57° C. for TFPI2and KISS1, and 58° C. for β-globin.

Statistical analyses were performed using the Sigma Stat 2.03 software(SPSS).

B. Results

Identification of Placental-specific Genes by High-densityOligonucleotide Array Analysis of Placental Tissues and Paired MaternalBlood Samples

Gene expression profiles of five first-trimester CVS and five termplacentas were obtained by microarray analysis of each individual tissuesample. A total of 7226 and 8871 gene transcripts were found to beexpressed in the CVS samples and term placentas, respectively. It hasbeen previously reported that circulating DNA in the plasma of normalindividuals is predominantly derived from hematopoietic cells (Lui etal., Clin. Chem., 48:421–427, 2002). Thus, it is hypothesized that muchof the background maternal nucleic acids in maternal plasma alsooriginate from the hematopoietic compartment. As the ultimate aim of thestudy was to identify placental-expressed transcripts that arefetal-specific amongst the circulating RNA molecules in maternal plasma,the present inventors further obtained the gene expression profiles ofpaired maternal whole blood and compared these profiles with those ofthe corresponding placental tissues using the GeneChip® Microarray Suite5.0 software (Affymetrix). Fetal-specific placental-expressedtranscripts in early pregnancy were identified by selecting transcriptswhose expression levels were “increased” in the CVS tissues whencompared to the corresponding whole blood samples in all fivecomparisons. Fetal-specific transcripts of late pregnancy were similarlyidentified from the term placentas when compared to the paired maternalwhole blood samples. After these procedures, transcripts that werehighly expressed in both the placental tissues and maternal blood cellswere eliminated, resulting in a panel of 1245 and 1743 transcriptsidentified for the first and third trimesters of pregnancy,respectively. The transcripts on these two panels were then sorted indescending order according to the medians of the five CVS or five termplacental tissue microarray expression signals (see Supplementary TablesA and B for the 50 most highly-expressed transcripts for early and latepregnancies, respectively). The two resultant panels consist ofcandidate transcripts that are potentially detectable in maternal plasmaas fetal-specific markers. The strategy used in the identification ofsuch fetal-specific markers is summarized in FIG. 1. The fact that threemRNA transcripts, namely, hPL, βhCG, and CRH, that had been previouslyfound detectable in maternal plasma are present in the list, providesindependent validation of this approach. However, as previous studieshave not included information on the gene expression levels in theplacenta and maternal plasma, these three transcripts were included infurther analysis, together with the three novel markers identified inthe list, namely, TFPI2, KISS1, and PLAC1. In order to compare therelative gene expression profiles between placental tissues and maternalplasma, transcripts located at different positions on the list wereselected. The median microarray expression signal intensities of thesesix transcripts are summarized in Table 1. The signal intensity of eachtranscript was first subjected to global scaling of the overallintensities of all arrays to a target intensity value of 500, and themedians of the scaled transcript intensities in the 5 CVS and 5 termplacental tissues were determined.

Development of Real-time QRT-PCR Assays for Measurement ofPlacental-expressed Transcripts in Maternal Plasma

Six one-step real-time QRT-PCR assays were used. The six selectedplacental mRNA transcripts were quantified in paired placental tissuesand plasma samples from 10 first and 10 third trimester pregnant womenby QRT-PCR. The six transcripts were detectable from the placentaltissues of all cases. The detectability and the median concentrations ofthese transcripts in both the first and the third trimester maternalplasma are summarized in Table 1. These results demonstrate that asignificant proportion of the selected placental-expressed genetranscripts identified by microarray analysis can indeed be detected inmaternal plasma. In general, transcripts with relatively higher medianmicroarray signal intensities are more readily detectable in maternalplasma. On the contrary, a median maternal plasma concentration of zerocopy is noted for the least abundant transcripts among the six studiedplacental-expressed genes, namely, PLAC1 in the first trimester, andhCGβ and PLAC1 in the third trimester. Hence, these data suggest thatplacental-expressed transcripts can be robustly detected in maternalplasma provided that the expression level exceeds a threshold microarraysignal.

Clearance of the Placental-expressed Transcripts from Maternal Plasmaafter Delivery

If the studied transcripts were pregnancy-specific, one would expectthat they would be cleared from maternal plasma after delivery. As shownin the previous studies (Ng et al., Proc. Natl. Acad. Sci. USA,100:4748–4753, 2003; and Ng et al., Clin. Chem., 49:727–731, 2003), hPLand CRH mRNA molecules were cleared rapidly from maternal plasma afterdelivery. To investigate the clearance of TFPI2, KISS1, and PLAC1 mRNAfrom maternal plasma, plasma samples from 10 pregnant women wereobtained before and at 24 hours after delivery. In the pre-deliveryplasma samples, the median TFPI2 and KISS1 mRNA concentrations were 112copies/ml and 88 copies/ml, respectively. Both transcripts were notdetected in any of the postpartum plasma samples (FIG. 8A and FIG. 8Bfor TFPI2 and KISS1 mRNA, respectively). For PLAC1 mRNA, the transcriptwas detected in 4 of the 10 pre-delivery plasma samples, while no signalwas detected in any of the postpartum samples (FIG. 8C). As a control,GAPDH mRNA was detected in all pre- and post-delivery plasma samples,with no systematic change in the concentrations (Wilcoxon test,P=0.563).

Validation of mRNA Analysis in Maternal Plasma for Non-invasivePlacental Gene Expression Profiling

Direct evidence was sought that the measurement of placental transcriptlevels in maternal plasma would provide an indirect measure of geneexpression levels in the placenta. Paired placental and plasma samplesfrom 10 first and 10 third trimester pregnancies were analyzed for hPL,hCGβ, CRH, TFPI2, KISS1, and PLAC1 mRNA by QRT-PCR. It was reasoned thatif the levels of these transcripts in maternal plasma were indeed areflection of their respective levels in the placenta, then a positivecorrelation should be seen between the relative concentrations of thesetranscripts in the placenta and in maternal plasma. One practicalapproach to express the relative levels of these transcripts would be tonormalize their levels with respect to a common placental-specifictranscript. As hPL mRNA could be detected throughout pregnancy (Ng etal., Proc. Natl. Acad. Sci. USA, 100:4748–4753, 2003), hPL mRNA waschosen as a reference. The normalized level for each transcript wascalculated by dividing the transcript level in an individual placenta orplasma sample to the corresponding hPL mRNA level in the same sample.The comparison was only made for transcripts that were detectable inmaternal plasma. As PLAC1 and hCGβ were not detectable at all in thecurrent series of first and third trimester maternal plasma samples,respectively, they were not included in the respective analyses. FIGS.9A and 9B show plots of the normalized transcript levels for hCGβ, CRH,TFPI2, KISS1, and PLAC1 mRNA in the placentas and the paired maternalplasma. A positive correlation was seen between the placental andmaternal plasma results for both the first (Spearman correlationanalysis, r=0.452, P<0.05) and third (Spearman correlation analysis,r=0.661, P<0.05) trimesters. As a control, two non-placental-specificmRNA transcripts were also analysed: β-globin for the third trimesterand GAPDH for both first and third trimester samples. As can be seen inFIGS. 9A and 9B, these non-placental-specific transcripts clearly didnot follow the correlative trend exhibited by the placental-specifictranscripts. These data suggest that the placental-specific mRNA levelsmeasured from maternal plasma may be used to assess placental geneexpression.

C. Conclusion

A strategy has been developed that allows the systematic identificationof a large panel of fetal-specific RNA markers in maternal plasma (FIG.7). This strategy was devised based on the previous finding that theplacenta is an important source of circulating fetal RNA in maternalplasma and the predominant haematological origin of plasma DNA in normalindividuals. High-density oligonucleotide microarrays were used tosystematically identify a large panel of placental-expressed transcriptsand to select the potentially fetal-specific transcripts for maternalplasma detection, with genes expressed by blood cells being discarded aspotential targets (Supplementary Tables A and B). Three previouslyidentified plasma placental-specific mRNA markers, coding for hPL, hCGβ,and CRH, were present in the target list and provided independentvalidation of the transcript selection strategy. In addition, three mRNAmarkers identified in the list, namely, TFPI2, KISS1, and PLAC1,previously unexplored for non-invasive prenatal monitoring, were alsodetectable in maternal plasma by QRT-PCR provided that their placentaltissue expression level was above a certain threshold. Their rapidclearance from the maternal plasma after delivery confirms theplacental-specificity. These findings add further weight to the validityof the strategy used for the identification of fetal-specifictranscripts in maternal plasma.

In contrast to the placental-specific mRNA species, no correlation wasobserved between the plasma and placental normalized mRNA levels ofnon-placental-specific transcripts, namely, GAPDH and β-globin. Comparedwith the placental-specific transcripts, relatively more GAPDH andβ-globin mRNA was present in the maternal plasma, which might beexplained by the contribution of such non-placental-specific transcriptsfrom maternal tissues, e.g., the hematopoietic cells.

In conclusion, it has been demonstrated that circulating placental mRNAin maternal plasma could be used for detection of pregnancy and fornon-invasive prenatal gene expression profiling. This study has furtheroutlined a microarray-based approach for rapidly and systematicallyidentifying new placental mRNA markers to be used for this purpose. Thisdevelopment has particular implications for the generation of newmarkers for the studying and monitoring of conditions known to beassociated with placental pathology, such as trisomy 21 andpre-eclampsia. In addition, the findings of this study may haveimplications beyond pregnancy-related diseases. For example, astumor-associated mRNA has been detected in the plasma/serum of cancerpatients (see, e.g., Lo et al., Clin. Chem., 45:1292–1294, 1999; andSilva et al., Gut., 50:530–534, 2002), a similar approach can be usedfor rapidly generating new plasma RNA-based tumor markers.

All patents, patent applications, and other publications cited in thisapplication are incorporated by reference in the entirety.

TABLE 1A Summary of microarray results in CVS samples and QRT-PCRresults in first-trimester maternal plasma of the six selected placentaltranscripts. median detectability Median concentration CVS in first- infirst- microarray trimester trimester plasma Transcript signal plasma(%) (copies/ml) βhCG 31857 90 4969 hPL 26503 90 769 TFPI2 24180 70 38KISS1 19756 50 18 CRH 6941 70 16 PLAC1 3020 0 0

TABLE 1B Summary of microarray results in term placental tissues andQRT-PCR results in third-trimester maternal plasma of the six selectedplacental transcripts. median term detectability median concentrationplacentas in third- in third- microarray trimester trimester plasmaTranscript signal plasma (%) (copies/ml) hPL 33972 100 14707 TFPI2 24896100 189 CRH 17077 70 98 KISS1 16033 100 50 βhCG 9759 0 0 PLAC1 9755 40 0

SUPPLEMENTARY TABLE A Microarray detection of the 50 most highlyexpressed genes in CVS tissues Transcript Probe Set ID GenBank acc.Signals (median) *chorionic gonadotropin, beta polypeptide 205387_s_atNM_000737.1 31857.3 placental lactogen (v.4) 208356_x_at NM_022642.128651.4 growth hormone 1 (v.5) 208068_x_at NM_022562.1 28500.7 growthhormone 1 (v.4) 208069_x_at NM_022561.1 27624.7 chorionicsomatomammotropin hormone 2 (v.1) 203807_x_at NM_020991.2 27513.2placental lactogen (clone MGC:14518) 211739_x_at BC005921.1 27185.9chorionic somatomammotropin hormone-like 1 (v.2) 208294_x_at NM_022578.127104.4 *placental lactogen (v.1) 202493_x_at NM_001317.2 26503.0chorionic somatomammotropin hormone 2 (v.3) 208342_x_at NM_022645.125820.6 placental lactogen (v.3) 208357_x_at NM_022641.1 25777.3chorionic somatomammotropin hormone 2 (v.4) 208341_x_at NM_022646.125603.1 pregnancy specific beta-1-glycoprotein 1 208257_x_at NM_006905.125453.4 growth hormone 1 (v.2) 206885_x_at NM_022559.1 24834.8 chorionicsomatomammotropin hormone-like 1 (v.1) 207285_x_at NM_001318.2 24796.9chorionic somatomammotropin hormone-like 1 (v.5) 208293_x_at NM_022581.124709.4 pregnancy specific beta-1-glycoprotein 6 209738_x_at M31125.124370.6 pregnancy specific beta-1-glycoprotein 3 203399_x_at NM_021016.124262.4 chorionic somatomammotropin hormone-like 1 (v.4) 208295_x_atNM_022580.1 24190.7 *tissue factor pathway inhibitor 2 209278_s_atL27624.1 24179.9 glycoprotein hormones, alpha polypeptide 204637_atNM_000735.2 23720.4 growth hormone 1 (v.3) 206886_x_at NM_022560.123475.7 pregnancy specific beta-1-glycoprotein 3 215821_x_at AB019570.123095.5 pregnancy specific beta-1-glycoprotein 3 211741_x_at BC005924.123025.1 growth hormone variant mRNA 211151_x_at AF185611.1 22792.2prostate differentiation factor 221577_x_at AF003934.1 22685.4 growthhormone 1 (v.1) 205840_x_at NM_000515.2 22446.6 chorionicsomatomammotropin hormone-like 1 (v.3) 205958_x_at NM_022579.1 22097.6pregnancy specific beta-1-glycoprotein 4 208191_x_at NM_002780.1 20730.7pregnancy specific beta-1-glycoprotein 2 208134_x_at NM_031246.1 20233.5pregnancy specific beta-1-glycoprotein 9 209594_x_at M34421.1 19939.4*KiSS-1 metastasis-suppressor 205563_at NM_002256.1 19755.8 placentallactogen (v.2) 206475_x_at NM_022640.1 19367.1 pregnancy specificbeta-1-glycoprotein 5 204830_x_at NM_002781.1 19182.6 growth hormone 2211508_s_at AF006060.1 18019.7 S100 calcium binding protein P 204351_atNM_005980.1 17970.2 pregnancy specific beta-1-glycoprotein 6 208106_x_atNM_002782.3 17566.8 chorionic somatomammotropin hormone 2 (v.2)207770_x_at NM_022644.1 17244.2 CD63 antigen (melanoma 1 antigen)200663_at NM_001780.1 17061.2 delta-like 1 homolog (Drosophila)209560_s_at U15979.1 16415.0 fibronectin 1 210495_x_at AF130095.116046.9 Homo sapiens cDNA: FLJ22066 fis, clone HEP10611 202409_at X0786816024.8 fibronectin 1 216442_x_at AK026737.1 15834.1 ribosomal proteinL31 200963_x_at NM_000993.1 15252.6 a disintegrin and metalloproteinasedomain 12 (v.2) 204943_at NM_021641.1 15007.3 collagen, type III, alpha1 (clone HEMBA1001071) 215076_s_at AU144167 14999.1 pregnancy specificbeta-1-glycoprotein 9 207733_x_at NM_002784.1 14819.7 secretedphosphoprotein 1 209875_s_at M83248.1 14665.3 Epstein-Barr virus inducedgene 3 219424_at NM_005755.1 14617.0 collagen, type III, alpha 1201852_x_at AI813758 14494.7 fibronectin 1 211719_x_at BC005858.114157.1 v, transcripts variant; * transcripts selected for QRT-PCR study*cortocotropin releasing hormone is located at position 156 on this list*placenta-specific 1 is located at position 412 on this list

SUPPLEMENTARY TABLE B Microarray detection of the 50 most highlyexpressed genes in term placental tissues Transcript Probe Set IDGenBank acc. Signals (median) chorionic somatomammotropin hormone 2(v.1) 203807_x_at NM_020991.2 35768.0 placental lactogen (clone MGC:1451) 211739_x_at BC005921.1 35732.4 placental lactogen (v.4)208356_x_at NM_022642.1 35099.3 *placental lactogen (v.1) 202493_x_atNM_001317.2 33972.4 growth hormone 1 (v.5) 208068_x_at NM_022562.133265.8 chorionic somatomammotropin hormone-like 1 (v.2) 208294_x_atNM_022578.1 32947.1 placental lactogen (v.3) 208357_x_at NM_022641.132826.0 chorionic somatomammotropin hormone 2 (v.4) 208341_x_atNM_022646.1 32231.7 chorionic somatomammotropin hormone 2 (v.3)208342_x_at NM_022645.1 31623.2 growth hormone 1 (v.4) 208069_x_atNM_022561.1 30524.9 chorionic somatomammotropin hormone-like 1 (v.4)208295_x_at NM_022580.1 30349.2 chorionic somatomammotropin hormone-like1 (v.5) 208293_x_at NM_022581.1 30076.9 growth hormone 1 (v.2)206885_x_at NM_022559.1 29888.7 pregnancy specific beta-1-glycoprotein 9209594_x_at M34421.1 29462.1 growth hormone 1 (v.1) 205840_x_atNM_000515.2 28856.8 pregnancy specific beta-1-glycoprotein 6 209738_x_atM31125.1 28736.3 chorionic somatomammotropin hormone-like 1, (v.1)207285_x_at NM_001318.2 28532.6 growth hormone 1 (v.3) 206886_x_atNM_022560.1 27576.2 pregnancy specific beta-1-glycoprotein 1 208257_x_atNM_006905.1 26521.2 pregnancy specific beta-1-glycoprotein 3 211741_x_atBC005924.1 26306.1 growth hormone variant (GHV) mRNA 211151_x_atAF185611.1 26127.2 pregnancy specific beta-1-glycoprotein 3 203399_x_atNM_021016.1 25987.8 placental lactogen (v.2) 206475_x_at NM_022640.125873.3 chorionic somatomammotropin hormone-like 1 (v.3) 205958_x_atNM_022579.1 25597.7 a disintegrin and metalloproteinase domain 12 (v.2)204943_at NM_021641.1 25592.8 pregnancy specific beta-1-glycoprotein 3215821_x_at AB019570.1 24944.4 *tissue factor pathway inhibitor 2209278_s_at L27624.1 24896.4 glycoprotein hormones, alpha polypeptide204637_at NM_000735.2 24831.5 pregnancy specific beta-1-glycoprotein 5204830_x_at NM_002781.1 23928.8 pregnancy specific beta-1-glycoprotein 2208134_x_at NM_031246.1 23786.0 pregnancy specific beta-1-glycoprotein 4208191_x_at NM_002780.1 23475.2 chorionic somatomammotropin hormone 2(v.2) 207770_x_at NM_022644.1 22227.9 pregnancy specificbeta-1-glycoprotein 9 207733_x_at NM_002784.1 22088.4 prostatedifferentiation factor 221577_x_at AF003934.1 20335.8 cytochrome P450,subfamily XIX 203475_at NM_000103.1 20147.5 delta-like 1 homolog(Drosophila) 209560_s_at U15979.1 19268.5 pregnancy specificbeta-1-glycoprotein 6 208106_x_at NM_002782.3 19225.4 Homo sapiens cDNAFLJ39399 fis, clone PLACE6011041 213332_at AL031290 18524.9 S100 calciumbinding protein P 204351_at NM_005980.1 18480.2 Homo sapiens cDNA:FLJ22066 fis, clone HEP10611 202409_at X07868 17518.8 CD59 antigenp18–20 200983_x_at NM_000611.1 17295.0 *corticotropin releasing hormone205630_at NM_000756.1 17076.6 a disintegrin and metalloproteinase domain12 (v.1) 202952_s_at NM_003474.2 16992.6 growth hormone 2 211508_s_atAF006060.1 16251.8 pregnancy specific beta-1-glycoprotein 7 205602_x_atNM_002783.1 16174.5 KiSS-1 metastasis-suppressor 205563_at NM_002256.116033.3 dipeptidylpeptidase 7 200878_at AF052094.1 15227.5hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-204515_at NM_000862.1 14394.6 isomerase 1 Homo sapiens mRNA full lengthinsert cDNA clone EUROIMAGE 201981_at AA148534 14277.2 1695532 fibulin 1201787_at NM_001996.1 14020.6 v, transcripts variant; * transcriptsselected for QRT-PCR study *placenta-specific-1 is located at position82 on this list *chorionic gonadotropin, beta polypeptide is located atposition 84 on this list

1. A method for diagnosing, or predicting preeclampsia in a pregnantwoman, the method comprising the steps of: (i) quantitativelydetermining the amount of mRNA encoding human corticotropin releasinghormone (hCRH) in the pregnant woman's plasma or serum; and (ii)comparing the amount of mRNA from step (i) to a standard controlrepresenting the amount of hCRH mRNA in the plasma or serum of anaverage non-preeclamptic pregnant woman, wherein an increase in theamount of mRNA compared with the standard control indicates preeclampsiaor an increased risk of developing preeclampsia.
 2. The method of claim1, wherein step (i) is performed by reverse transcriptase polymerasechain reaction (RT-PCR).
 3. The method of claim 1, wherein step (i) isperformed by a polynucleotide hybridization method.
 4. The method ofclaim 1, wherein the woman is during the first trimester of gestation.5. The method of claim 1, wherein the woman is during the second orthird trimester of gestation.
 6. The method of claim 1, wherein thepregnant woman's plasma is used in step (i).
 7. The method of claim 1,wherein the pregnant woman's serum is used in step (i).
 8. The method ofclaim 1, wherein the increase in the amount of mRNA from the standardcontrol is more than 2-fold.